Release of intracellular material

ABSTRACT

Intracellular material is released from bacterial, yeast, plant, animal, insect or human cells by the application of a low voltage such as 1 to 10 V to a suspension containing the cells. The conditions may be selected such that DNA released from the cells is electrochemically denatured so as to be available for use in an amplification procedure.

The present invention relates to methods for producing release ofintracellular material from cells.

Current methods for cell lysis and isolation of cellular material arelaborious and time consuming and require a number of steps. For example,the preparation of DNA from bacteria requires a protocol of no fewerthan ten individual steps. To produce effective cell lysis in gramnegative bacteria, treatment with reagents such as EDTA and digestionwith enzymes such as lysosyme and RNase are required. This is followedby cold shock, osmotic shock or boiling in order to release cellularmaterial. Multiple steps are then necessary to harvest nucleic acidsfrom the lysed preparation.

In the case genomic DNA isolation these are, following lysis of cells torelease the DNA:—

-   -   digestion of RNA and proteins with enzymes,    -   removal of contaminants, usually by solvent extraction,    -   and finally, dialysis or ethanol precipitation steps to give a        clean preparation.

Plasmid extraction comprises cell lysis and selective precipitation ofgenomic DNA followed by the purification of plasmid DNA by gradientcentrifugation or by ion-exchange chromatography. DNA extractiontechniques currently in use include phenol-chloroform extraction andsalting out methods for genomic DNA, and cesium chloride/ethidiumbromide density gradients, and ion-exchange columns, for plasmid DNA. Inaddition kits are widely used for purification of DNA and DNA fragmentswhich are based on the precipitation of DNA under chaotropic conditions.All of these techniques have their limitations. For example, densitygradients and phenol/chloroform extraction are time consuming processesto use, taking up to 24 hours to perform. In addition, the copious useof phenol for these purposes is highly undesirable due to its toxic andcaustic nature. Methods of isolating large fragments of DNA can resultin DNA shearing.

It is known that electroporation using voltages in the kilovolt rangecan produce release of intracellular material through the permeablisedcell membrane produced transiently in the electroporation process; seefor instance Brodelius P. E. Funk C, Shillito R. D., Plant Cell Reports7, 186 (1988) and Heery D. M., Powell R., Gannon, F., and Dunican L. K.Nuc. Acids Res. 17, 10131 (1989).

Electroporation involves the application of high voltages, typically inexcess of 1 kV in pulses of short duration in the order of milliseconds.Generally, the field gradient between the electrodes across which thevoltage is applied to a suspension containing the cells to beelectro-porated will be in excess of 1 kV per cm. This requiressophisticated and expensive apparatus.

It has now been found that it is possible to obtain release ofintracellular material from cells by the application of voltages of alower order of magnitude not previously thought to be capable ofaffecting cell membrane structure in such a way.

Accordingly, the present invention provides a method of producingrelease of intracellular material from cells comprising applying avoltage of not more than 50 volts to a suspension of said cells.

Preferably, the voltage is from 0.5 to 50 volts with a strong preferencefor voltages in the lower part of this range e.g. from 0.5 to 15 volts,most preferably from 1 to 10 volts.

The voltage may be a DC voltage or an AC voltage.

Unlike the practice in electroporation, the voltage may be appliedcontinuously, subject to avoiding excess heating effects which maybecome a problem if the voltage is in excess of 15 volts. Preferably thevoltage is applied for a period of at least 30 seconds, more preferablyfor at least 2 minutes, e.g. from 2 to 20 minutes. Preferably thevoltage is applied continuously for a period as specified above, butthis process may be repeated, e.g. by the application of voltage forrepeated periods of several seconds to several minutes, e.g. 5 secondsto 10 minutes.

The electrodes by which the voltage is applied may preferably be spacedby 10 mm or less, e.g. 5 to 7 mm.

However, it may be preferred to optimise the conditions for producingdenaturation of double-stranded DNA released from the cells, in whichcase a smaller electrode spacing will be desirable. To accomplishdenaturation of released DNA, preferably the voltage is applied to thesuspension between closely spaced electrodes, preferably not spaced bymore than 5 mm at their closest approach, e.g. by no more than 1.5 mmand most preferably by no more than 0.5 mm.

One of the electrodes may be constituted by a container of conductivematerial in which the sample being treated is contained.

The process may be conducted to produce cell lysis and to produce therelease of intracellular materials including proteins and nucleic acids,including double stranded DNA, and other biomolecules. A process forproducing denaturation of double-stranded nucleic acid utilisingapparatus suitable for use in the present invention is described inApplication PCT/GB9S/00542. This process is itself an improvement onprocesses for electrochemical denaturation of double-stranded nucleicacid described in WO92/04470 and WO93/15224. As disclosed in thosespecifications, nucleic acid may be denatured reversibly by theapplication of an electrical voltage and such denaturation may be usedas a step in a number of more complex tasks including hybridisationstudies and nucleic acid amplification procedures such as PCR.

Nucleic acids released from cells by methods according to the presentinvention may be further processed according to the teachings of thesespecifications.

Accordingly, the present invention includes a method of producingsingle-stranded nucleic acid which comprises releasing double strandednucleic acid from cells by applying a voltage of not more than 50 voltsto a suspension of said cells with an electrode to release nucleic acidfrom said cells and denaturing the double-stranded nucleic acid byapplying the same or a different voltage to said suspension with saidelectrodes to convert said double-stranded nucleic acid tosingle-stranded nucleic acid.

The range of voltage within which production of denaturation in this wayis achievable will not be as wide as the range of voltage appropriatefor producing cell lysis and accordingly it is preferred that in thedenaturation stage, a voltage of from 0.5 to 3 volts is applied, morepreferably from 1.5 to 2.5 volts, measured as a voltage differencebetween the electrodes.

As described in WO92/04470, one may employ a promoter compound such asmethyl viologen to produce more rapid denaturation.

More generally, the promoter may be any inorganic or organic moleculewhich increases the rate or extent of denaturation of the double helix.It should be soluble in the chosen reaction medium. It preferably doesnot affect or interfere with DNA or other materials such as enzymes oroligonucleotide probes which may be present in the solution.Alternatively, the promoter may be immobilised to the electrode orincluded in material from which the electrode is constructed. It may bea water soluble compound of the bipyridyl series, especially a viologensuch as methyl viologen or a salt thereof. Whilst the mechanism ofoperation of such promoters is not presently known with certainty, it isbelieved that the positively charged viologen molecules interact betweenthe negatively charged nucleic acids such as DNA and the negativelycharged cathode to reduce electrostatic repulsion therebetween and henceto promote the approach of the DNA to the electrodes surface where theelectrical field is at its strongest. Accordingly, we prefer to employas promoters compounds having spaced positively charged centres, e.g.bipolar positively charged compounds. Preferably the spacing between thepositively charged centres is similar to the spacing between thepositively charged centres in viologen. Other suitable viologens includeethyl viologen. isopropyl viologen and benzyl viologen.

Other promoters are described in WO93/15224, i.e. multivalent cationssuch as magnesium. Other multivalent cations which are effective andwhich can be used include lanthanum (La³⁺). The cations as the promotermay include inorganic cations complexed with inorganic or organicligands, e.g. Pt(NH₃)₆ ⁴⁺ and Cr(NH₃)₆ ²⁺. The method of release ofintracellular material of the present invention may be practised in thepresence or in the absence of such a promoter, or in the presence orabsence of a release promoting amount of any promoter.

At least in the denaturation stage it is preferred that where theelectrodes most closely approach one another, one or both of theelectrodes is pointed. Such an electrode may be provided with a singlepoint or a plurality of points. There appears to be someinter-relationship between the ideal voltage applied and the shape ofthe electrode and it may be that there is a preferred or ideal fieldgradient at the point of the electrode which can be achieved byadjustment of the voltage to suit the sharpness of the part of theelectrode at which the denaturation takes place. Optionally, one canconduct the denaturation using a constant current supply rather than aregulated voltage and this may serve to compensate for variations in thegeometrical set-up of the electrodes between different denaturationoperations.

Where a constant current regime is employed, it will generally bepreferable to use a current of from 80 to 160 μA, e.g. about 100 to 125μA.

Optionally, the process may be conducted using a three electrode systemof the kind described in WO92/04470 but generally it is preferred thatthe volume of solution employed according to this invention is smalle.g. 1 ml or less, preferably very small e.g. 100 μl or less, e.g. about25 μl to 40 μl. When using very small reaction volumes of this kind, itwill generally not be practical to use a three electrode system.

The processes e.g. of cell lysis and of denaturation ma % each becarried out at ambient temperatures or if desired at temperatures up toadjacent the pre-melting temperature of the nucleic acid. Each processmay be carried out at a pH of from 3 to 10, conveniently about 7.Generally, more rapid denaturation is obtained at lower pH. For somepurposes therefore a pH somewhat below neutral e.g. about pH 5.5 may bepreferred. The cells may be suspended in an aqueous solution containinga buffer whose nature and ionic strength are such as not to interferewith the strand separation process.

Preferably, the solution contains a buffer at a concentration at least10 mM e.g. about 25 mM. Optionally, the solution may contain furthersalts such as magnesium chloride and sodium chloride. Preferably, themethod is conducted in a buffer of the kind used in PCR or in LCRprocedures.

Preferably, therefore the ionic strength of the solution is above 20 mM,e.g. 25 to 50 mM.

The release of nucleic acid and the denaturing process according to theinvention may be incorporated as steps in a number of more complexprocesses, e.g. procedures involving the analysis and/or theamplification of nucleic acid. Some examples of such processes aredescribed below.

We have found that by virtue of the superior electrochemical cell designdescribed in PCT/GB95/00542 it is possible to achieve denaturationwithin less than 3 minutes e.g. from 1 to 2 minutes or less even in thepresence of materials such as PCR buffers.

This makes it possible to practise a process of repeated denaturation ofdouble-stranded nucleic acid wherein the nucleic acid is denatured by aprocess as described above in which the voltage is applied as sequenceof repeated pulses having a duration of up to 2 minutes, preferably upto only 1 minute. Between pulses, the voltage may be turned off orreversed for a period which is preferably equal to the period for whichthe voltage is applied. It is possible to employ pulses of considerablyhigher frequencies than described above, e.g. from 1 to 100 Hz.Depending upon the purpose for which the denaturation is beingconducted, it may not be necessary to achieve any substantial amount ofconversion of double-stranded to single-stranded nucleic acid in eachdenaturation cycle. It may be sufficient merely to initiate denaturationelectrochemically. For instance, in an amplification procedure, ifsufficient denaturation occurs to allow binding of a primer, theextension of the primer by nuclease may be relied upon to displace theunprimed strand of the original nucleic acid from its binding partnerover: the remainder of the length of the nucleic acid.

The invention further provides a process of amplifying a target sequenceof nucleic acid comprising hybridisation, amplification and denaturationof nucleic acid wherein the nucleic acid is released from a cell asdescribed above and said denaturation is conducted by subjecting asolution containing said nucleic acid to a voltage applied betweenelectrodes for a period of up to 2 minutes under conditions such as tocovert at least a portion of the nucleic acid to a wholly or partiallysingle-stranded form in the solution.

Preferably, the electrode configuration used in such a process is asdescribed above. Preferably, the voltage is applied as a repeating pulsehaving a duration of up to 1 minute but preferably shorter, e.g. up to0.1 minute or even much shorter, e.g. at 1 to 100 Hz.

Preferably, the amplification procedure is PCR or LCR.

Thus the present invention includes a process for replicating a nucleicacid which comprises: releasing double stranded nucleic acid from cellsby a process as described above; separating the strands of a sampledouble-stranded nucleic acid in solution under the influence of anelectrical voltage applied to the solution from an electrode;hybridising the separated strands of the nucleic acid with at least oneoligonucleotide primer that hybridises with at least one of the strandsof the denatured nucleic acid; synthesising an extension product of theor each primer which is sufficiently complementary to the respectivestrand of the nucleic acid to hybridise therewith; and separating the oreach extension product from the nucleic acid strand with which it ishybridised to obtain the extension product.

The replication process may be a step in a 3SR or NASBA amplificationprocedure or a strand displacement assay.

In such a polymerase mediated replication procedure, e.g. a polymerasechain reaction procedure, it may not be necessary in all cases to carryout denaturation to the point of producing wholly single-strandedmolecules of nucleic acid. It may be sufficient to produce a sufficientlocal and/or temporary weakening or separation of the double helix inthe primer hybridisation site to allow the primer to bind to its target.Once the primer is in position on a first of the target strands,rehybridisation of the target strands in the primer region will beprevented and the other target strand may be progressively displaced byextension of the primer or by further temporary weakening orseparation-processes.

Preferably, the said replication process further comprises repeating theprocedure defined above cyclicly, e.g. for more than 10 cycles, e.g. upto 20 or 30 cycles. In the amplification process the hybridisation stepis preferably carried out using two primers which are complementary todifferent strands of the nucleic acid.

The denaturation to obtain the extension products as well as theoriginal denaturing of the target nucleic acid is preferably carried outby applying to the solution of the nucleic acid the voltage from theelectrodes.

The process may be a standard or classical PCR process for amplifying atleast one specific nucleic acid sequence contained in a nucleic acid ora mixture of nucleic acids wherein each nucleic acid consists of twoseparate complementary strands, of equal or unequal length, whichprocess comprises:

-   -   (a) treating the strands with two oligonucleotide primers, for        each different specific sequence being amplified, under        conditions such that for each different sequence being amplified        an extension product of each primer is synthesised which is        complementary to each nucleic acid strand, wherein said primers        are selected so as to be substantially complementary to        different strands of each specific sequence such that the        extension product synthesised from one primer, when it is        separated from its complement, can serve as a template for        synthesis of the extension product of the other primer;    -   (b) separating the primer extension products from the is        templates on which they were synthesised to produce        single-stranded molecules by applying the voltage from the        electrode to the reaction mixture; and    -   (c) treating the single-stranded molecules generated from        step (b) with the primers of step (a) under conditions such that        a primer extension product is synthesised using each of the        single strands produced in step (b) as a template.

Alternatively, the process may be any variant of the classical orstandard PCR process, e.g. the so-called “inverted” or “inverse” PCRprocess or the “anchored” PCR process.

The invention therefore includes the use of an amplification process asdescribed above in which a primer is hybridised to a circular nucleicacid released from a cell as described and is extended to form a duplexwhich is denatured by the application of the denaturing voltage, theamplification process optionally being repeated through one or moreadditional cycles.

The process of the invention is applicable to the ligase chain reaction.Accordingly, the invention includes a process for amplifying a targetnucleic acid comprising the steps of releasing the target nucleic acidfrom a cell as described followed by:

-   (a) providing nucleic acid of a sample as single-stranded nucleic    acid;-   (b) providing in the sample at least four nucleic acid probes,    wherein:    -   i) the first and second said probes are primary probes, and the        third and fourth of said probes are secondary nucleic acid        probes;    -   ii) the first probe is a single strand capable of hybridising to        a first segment of a primary strand of the target nucleic acid;    -   iii) the second probe is a single strand capable of hybridising        to a second segment of said primary strand of the target nucleic        acid;    -   iv) the 5′ end of the first segment of said primary strand of        the target is positioned relative to the 3′ end of the second        segment of said primary strand of the target to enable joining        of the 3′ end of the first probe of the 5′ end of the second        probe, when said probes are hybridised to said primary strand of        said target nucleic acid;    -   v) the third probe is capable of hybridising to the first probe;        and    -   vi) the fourth probe is capable of hybridising to the second        probe; and-   (c) i) hybridising said probes with nucleic acid in said sample;    -   ii) ligating hybridised probes to form reorganised fused probe        sequences; and    -   iii) denaturing DNA in said sample by applying a voltage to the        reaction mixture.

The electrochemical DNA release and amplification technique can be usedanalytically to detect and analyse a very small sample of DNA e.g. asingle copy gene in an animal cell or a single cell of a bacterium.

The temperature at which the process is carried out may be chosen tosuit whichever enzyme is used. Thus where Taq is used as polymerase, atemperature of 55 to 68° C. is preferred. If Klenow polymerase is used,ambient temperature will be suitable. It may be desirable to employknown protein stabilisation techniques to avoid electrical damage to thepolymerase, especially where a mesophyllic polymerase is used.

The invention includes a process for detecting the presence or absenceof a predetermined nucleic acid sequence in a cell which comprises:releasing nucleic acid from the cell as described, denaturing releaseddouble-stranded nucleic acid by means of a voltage applied to thenucleic acid; hybridising the denatured nucleic acid with anoligonucleotide probe for the sequence; and determining whether the saidhybridisation has occurred.

Thus, the invented process has application in DNA and RNA hybridisationwhere a specific gene sequence is to be identified e.g. specific to aparticular organism or specific to a particular hereditary disease ofwhich sickle cell anaemia is an example. To detect a specific sequenceit is first necessary to prepare a sample of DNA, by the release of theDNA from a cell as described which is in native double-stranded form. Itis then necessary to convert the double-stranded DNA to single-strandedform before a hybridisation step with a labelled nucleotide probe whichhas a complementary sequence to the DNA sample can take place. Theprocess of the invention can be used for this purpose in a preferredmanner by carrying out the following steps:

-   -   releasing DNA from a cell by the method described above;    -   denaturing the DNA by applying a voltage by means of an        electrode configuration as described to the sample DNA with        optionally a promoter in solution or bound to or part of the        structure of the electrode;    -   hybridising the denatured DNA with a directly labelled or        indirectly labelled nucleotide probe complementary to the        sequence of interest; and    -   determining whether the hybridisation has occurred, which        determination may be by detecting the presence of the probe, the        probe being directly radio-labelled, fluorescent labelled,        chemiluminescent labelled or enzyme-labelled or being an        indirectly labelled probe which carried biotin for example to        which a labelled avidin or avidin type molecule can be bound        later.

In a typical DNA probe assay it is customary to immobilise the sampleDNA to a membrane surface which may be composed of neutral or chargednylon or nitrocellulose. The immobilisation is achieved by chargeinteractions or by baking the membrane containing DNA in an oven. Thesample DNA can be heated to high temperature to ensure conversion, tosingle-stranded form before binding to the membrane or it can be istreated with alkali once on the membrane to ensure conversion to thesingle-stranded form. The disadvantages of such methods are:

-   -   heating to high temperature to create single-stranded DNA can        cause damage to the sample DNA itself;    -   the use of alkali requires an additional step of neutralisation        before hybridisation with the labelled probe can take place.

One improved method for carrying out DNA probe hybridisation assays isthe so-called. “sandwich” technique where a specific oligonucleotide isimmobilised on a surface. The surface having the specificoligonucleotide thereon is then hybridised with a solution containingthe target DNA in a single-stranded form, after which a second labelledoligonucleotide is then added which also hybridises to the target DNA.The surface is then washed to remove unbound labelled oligonucleotide,after which any label which has become bound to target DNA on thesurface can be detected later.

This procedure can be simplified by using the cell lysis and denaturingprocess of the invention to denature the target DNA from double-strandedinto the required single-stranded DNA which can hybridise to theimmobilised oligonucleotide. The working electrode, counter-electrodeand optionally a reference electrode and/or a promoter can beincorporated into a test surface or a well in which the DNA probe assayis to be carried out. The cell sample and oligonucleotide probes canthen be added and the voltage applied to release and denature the DNA.The resulting single-stranded DNA is hybridised with the specificoligonucleotide immobilised on the surface after which the remainingstages of a sandwich assay care carried out. All the above steps cantake place without a need for high temperatures or addition of alkalireagents as in the conventional process.

The release of the nucleic acids from the cell or cells and thedenaturation of the nucleic acids may be conducted under similarconditions in which case there may be no clear division between thesteps of cell release and denaturation. The nucleic acid may bedenatured as it is released from the cells.

Other cell contents than nucleic acid may of course be releasedaccording to the methods of the invention and the release of such othermaterials may be utilised in various ways for instance, the release ofspecific proteins may be used as a step in an assay procedure in whichsuch proteins are detected. RNA released may be detected inhybridisation assays or amplified, e.g. by 3SR or NASBA techniques.Generally, intracellular materials released by methods according to theinvention may be utilised for all of the purposes for which suchmaterials have been utilised when released by conventional cell lysisprocedures.

The invention will be further described and illustrated with referenceto the accompanying drawings in which:—

FIG. 1 is a cross-sectional view through an electrical cell for use inaccordance with the invention;

FIG. 2 is a gel produced in Example 1 showing release of protein fromcells;

FIG. 3 is a gel produced in Example 2 showing release of protein fromcells;

FIG. 4 is a gel produced in Example 3 showing PCR amplification of DNAreleased from cells; and

FIG. 5 is a cross-sectional view through an alternative electrical cellfor use in the invention.

The cell shown in FIG. 1 comprises a glass container 10 having twoelectrodes 14 dipping into a cell suspension 12 therein. The electrodes14 are parallel pointed carbon rods spaced by 5 to 8 mm and having adiameter above said tapered points of about 1 mm. A length of 5 to 15 mmof electrode is dipped into the suspension.

An alternative apparatus is shown in FIG. 5 where the cell illustratedcomprises a graphite block 10, containing a 4 mm diameter well 12 on itsupper surface, constituting a first electrode. A second electrode formedfrom a 2 mm diameter graphite rod 14 having a tapering end portion 16 ispressed into the well through an insulating collar of plastics material18. The rod is adjusted down-wards in the well until it forms a shortcircuit and is then lifted back by as little as possible to open thecircuit again. The capacity for liquid for the cell is approximately 25μl. A DC voltage is applied to the apparatus with the well being madepositive with respect to the rod by preferably 1.6 to 2.5 volts.

Although the rod electrode shown in FIG. 1 is blunt ended, it provides asharp edge between its flat end and its tapering frustoconical surface.An optional alternative configuration is for the rod to be sharpened toa point. This may be achieved by using a conventional pencil sharpener.The resulting electrode may be further smoothed using a blade orabrasive. Using such a pointed electrode, the capacity for liquid of thewell is increased to 40 μl.

Multiple processes according to the invention may be carried outsimultaneously in apparatus containing a multiplicity of samplereceiving wells each provided with a respective electrode pair, oneelectrode in each case optionally being the well itself. In a preferredform for such apparatus a block containing the wells has a liftable lidwith electrodes depending therefrom into the wells. Each well may have apair of electrodes on the lid in various possible electrodeconformations such as parallel rods, parallel plates, optionally ofmesh, or coaxial hollow cylinders, again optionally of mesh.Alternatively, single electrodes may be provided on the lid for eachwell and the block containing the wells may be conductive and may serveas a common second electrode. The block of wells may also containrespective electrodes for each well.

The lid may comprise a flat plate portion bearing the electrode orelectrodes for each well and a separate backing member bearingelectrical connections and circuitry which connects up the electrodeswhen the two parts are assembled. A single electrical supply to the unitmay be split by said circuitry and supplied in a controlled manner tothe electrodes such that each electrode is controlled, e.g. to aconstant voltage or constant current. The plate portion carrying theelectrode array may thereby be replaceable without replacement of thecontrol circuitry and may be made disposable. The plate portion and thebacking member may be aligned with one another on assembly by locatingpins and apertures and may similarly be aligned with the blockcontaining the wells, which also may be disposable.

The following example illustrate methods according to the invention.

EXAMPLE 1

Gram negative bacteria (E. Coli strain DH5αF (Gibco, BRL)) were grownovernight in Luria Bertani medium. The average cell density achieved was1/10⁹ cells ml⁻¹.

Harvested cells were washed in 1M Tris buffer, pH 8.0 and resuspended in⅕ of the volume of the original culture. Two carbon electrodes wereplaced in the sample as shown in FIG. 1 and direct voltages of 2 to 8Vwere applied for 2 to 5 minutes. Positive control samples were boiledfor 5 minutes. The cell debris was pelleted by centrifugation at 5000rpm for 5 minutes and 20 μl of the supernatant was analysed byeleccrophoresis on a polyacrylamide gel, stained with Coomassie blue.The data demonstrate that applying a potential difference of 8 volts and4 volts for 5 minutes (d.c.) resulted in protein release from the cell.Similar results are obtained using alternating voltage of 2 to 8V.

EXAMPLE 2

The same experimental conditions were used as in Example 1 above butusing reduced electrocution times at 8 volts. This resulted in therelease of protein from the cell even after only 1 minute.

EXAMPLE 3

In order to demonstrate DNA release and to increase the sensitivity ofdetection we assessed cell lysis by amplifying DNA using the polymerasechain reaction.

Escherichia coli, strain DH5αF (Gibco, BRL) were transformed with pBR322(Sigma) and grown overnight in Luria Bertani medium containing 100μgml⁻¹ ampicillin. The average cell density achieved was 1×10⁸ cellsml⁻¹. Cells were harvested by centrifugation at 10000 rpm for fiveminutes and washed in PCR buffer (10 mM Tris, 50 mM KCl, 2.5 mM MgCl₂,pH 8.3). Cells were resuspended in PCR buffer at a concentration fivetimes that of the original. Positive control samples were treated byboiling for 5 minutes.

Two carbon probe electrodes were placed into the sample and 4-8 V (d.c.)was applied (power supply; Thurlby 30V, 2A) for between 0.5 to 2minutes. The cell debris was pelleted and supernatants were analysed byPCR. PCR conditions were as follows; 0.1 μl/ml of sample in PCR buffer(as above), 1 μM (each) of primers ATGCGTCCGGCCGTAGAGGAT andGTATCACGAGGCCCTT, 200 μM of each of DATP, dCTP, dGTP, dTTP, SU/mlAmpliTaq DNA polymerase (Perkin Elmer). All reagent concentrations aregiven as the final concentration in a reaction volume made up with PCRbuffer (as above). Amplified DNA was analysed on agarose gels stainedwith ethidium bromide. An amplified DNA fragment of the expectedmolecular weight (417 bp) was observed in samples which had beensubjected to the shortest test time of 30 seconds (see FIG. 3). Thedensity of the bands indicated that cell lysis, induced by an appliedvoltage, released DNA in excess of the background (non-lysed cellscontrol) level.

1-17 (cancelled)
 18. A process of amplifying a target sequence ofnucleic acid comprising denaturation, hybridisation, and replication ofnucleic acid wherein the nucleic acid is released from a cell by amethod comprising applying a voltage of not more than 50 volts to asuspension containing said cell or cells, and said denaturation isconducted by subjecting a solution containing said nucleic acid to avoltage applied between electrodes for a period of up to 2 minutes underconditions such as to convert at least a portion of the nucleic acid toa wholly or partially single-stranded form in the solution.
 19. Anamplification process as claimed in claim 18, wherein the amplificationprocedure is PCR or LCR.
 20. A process for replicating a nucleic acidwhich comprises: releasing double stranded nucleic acid from cells by amethod comprising applying a voltage of nor more than 50 volts to asuspension containing said cell or cells, separating the strands of asample double-stranded nucleic acid in solution under the influence ofan electrical voltage applied to the solution from an electrode;hybridising the separated strands of the nucleic acid with at least oneoligonucleotide primer that hybridises with at least one of the strandsof the denatured nucleic acid; synthesising an extension product of theor each primer which is sufficiently complementary to the respectivestrand of the nucleic acid to hybridise therewith; and separating the oreach extension product from the nucleic acid strand with which it ishybridised to obtain the extension product. 21 (canceled)